This invention relates to heat shock proteins of the Hsp60 family from Candida glabrata and Aspergillus fumigatus and a heat shock protein of the Hsp70 family from Neisseria meningitidis, including fragments thereof, and uses of such proteins and nucleic acid molecules encoding these proteins.
Meningitis is an infection of the fluid of the spinal cord and the fluid that surrounds the brain. The disease is caused either by a viral or a bacterial infection. Viral meningitis is typically less severe than bacterial meningitis and resolves without specific treatment. In contrast, bacterial meningitis can be rather severe and can cause brain damage, hearing loss or learning disability. Symptoms of meningitis are high fever, headache and stiff neck. These symptoms may develop over a span of several hours, or may take 1-2 days. Other symptoms may be nausea, vomiting, discomfort looking into bright light, confusion or sleepiness. In young children, the classical symptoms may be absent or may not be easily detected, and the child may appear to be slow, inactive, irritable, vomiting or feeding poorly. As the disease progresses, seizures may occur.
Bacterial meningitis may be caused by Haemophilus influenzae, Streptococcus pneumoniae or Neisseria meningitidis. Because all children (in the U.S.) are now given a vaccine against Haemophilus influenzae in the course of their routine immunizations, meningitis due to this organism is now relatively uncommon. Thus, the major bacterial disease-causing agents are now Streptococcus pneumoniae and Neisseria meningitidis. 
Early diagnosis and treatment are critically important. It must be determined whether symptoms are due to a viral or bacterial agents, and, if they are caused by a bacterial agent, which bacterium is involved. Present methods of diagnosis are relatively slow. They involve obtaining spinal fluid by performing a spinal tap. Bacteria are identified by cultivation of spinal fluid.
Bacterially-caused meningitis can be treated by antibiotics. However, it is critically important that treatment commence early in the course of the disease. Obviously, antibiotic therapy may be jeopardized by the development of antibiotic-resistant strains of disease-causing bacteria. Because of this concern, and also because of cost-benefit considerations, vaccination against the bacteria causing the disease would be preferable, at least in regions, in which the disease is endemic. As discussed before, U.S. children are routinely vaccinated against Haemophilus influenzae, but not against Neisseria meningitidis and Streptococcus pneumoniae. One such vaccine protects against four strains of Neisseria meningitidis. However, the vaccine appears not to be effective in children under 18 months of age. Similarly, a vaccine containing polysaccharide antigens for 14 of the 83 capsular types of Streptococcus pneumoniae was developed. The vaccine was found to be 57% effective in two large studies. As with the Neisseria vaccine, children under the age of two years do not appear to be protected by the vaccine. Thus, there is a need for improved vaccine against the latter two species of bacteria. The information provided here was obtained from publications by the Division of Bacterial and Mycotic Diseases of the National Center for Infectious Diseases, and the Centers for Disease Control and prevention, by Lonks and Medeiros, Antimicrobial Therapy 1 79:523-535, 1995, by Butler et al., JAMA 270:1826, 1993, and by Gotschlich et al., Antibodies in Human Diagnosis and Therapy 391-402 (Haber and Krause eds., 1977).
Aspergillosis is an opportunistic infection occurring in compromised individuals and is caused by molds of the genus Aspergillus, of which Aspergillus fumigatus is an important species. Aspergillus is ubiquitous and is distributed worldwide. Infection generally involves inhalation of fungal elements. Aspergillosis has several clinical manifestations, including colonization of the ear or the lungs, allergic pulmonary involvement, and invasive pulmonary and disseminated infections. The pulmonary invasive and disseminated forms of infections have a grave prognosis, including a high rate of mortality. Susceptible hosts include cancer patients, patients treated with immunosuppressive or cytotoxic drugs, and those otherwise debilitated such as AIDS patients, neutropenic cancer patients, or patients receiving adrenal corticosteroid drugs. Segal, Vaccines against Fungal Infections, CRC Crit. Rev. Microbiology 14:229, 1987. Rolston and Bodey, Infections in patients with cancer, Cancer Medicine (Holland et al. eds), 1997. The true incidence of aspergillosis is not known in the HIV/AIDS population, in part because the condition is frequently not diagnosed. However, it is clear that the incidence is increasing. Ampel has reported that more than 75 cases have been documented in the literature. Ampel, Emerging disease issues and fungal pathogens associated with HIV infection, Emerging Infectious Diseases 2:109-116, 1996. Aspergillosis has been reported to occur in 20-50% of patients with acute leukemia. It is noted that many cases of Aspergillus infection in cancer patients are not diagnosed until an autopsy is performed after death. Clearly, aspergillosis is becoming increasingly common among neutropenic patients and in cancer patients receiving corticosteroid drugs. Rolston and Bodey, supra. An article in 1992 by Bodey et al. reports on the incidence of fungal infections based on an international autopsy survey. Bodey et al., Fungal Infections In Cancer Patients, An International Autopsy Survey, Eur. J. Clin. Microbiol. Infect. Dis. 11:99-109, 1992. Countries included are Austria, Belgium, Canada, Germany, Italy, Japan, Netherlands and the UK. It was concluded that 25% of leukemia patients had fungal infections. Of these infected patients, 66% had candidiasis, and 34% aspergillosis. Estimates of rates of fungal infections in organ transplant patients as high as about 40% were published. Paya, Clin. Infect. Dis. 16:677-688, 1993. More than 80% of these infections were due to Candida and Aspergillus. As alluded to before, diagnosis of aspergillosis is not infrequently missed, and there is therefore a need for improved methods of diagnosis.
Candidiasis is a fungal infection caused by yeasts of the genus Candida. Among the more than 80 known species, only seven species appear to be pathogenic. The major disease-causing species is Candida albicans. Among the pathogenic species s also found Candida glabrata, formerly known as Torulopsis glabrata. Clinical manifestations of Candida infection range from superficial cutaneous infections to disseminated disease. Infections range from acute to chronic. They can involve skin and nails, the mucosal membranes of the mouth and vagina, and various internal organs such as the lungs, gastrointestinal tract, and circulatory and central nervous systems. Manifestations can be oral thrush, vaginitis, balanitis, diaper rash, chronic mucocutaneous conditions, bronchitis or pneumonia, meningitis, endocarditis, and septicemia. While the superficial forms of candidiasis have been well known since antiquity, the incidence of the disseminated forms has increased recently, presumably because of the extensive use of antibiotics, corticosteroids, cytotoxic drugs, organ transplantation and other complex surgical procedures. It is important to note that today the majority of systemic or invasive fungal infections are due to Candida species. Segal, supra. Stringer, Mass. High Tech. 14:3, 1997.
Mortality from systemic candidiasis remains high, in the order of 38-59%. The mainstay for treatment is amphotericin B, and an alternative is fluconazole. In a multicenter trial, amphotericin B was 79% effective, and fluconazole 70%. Note that Candida glabrata is resistant to fluconazole. Because mortality remains high, an effective vaccine against candidiasis to be used in high-risk populations would be desirable.
Since Candida albicans is the major cause of candidiasis, essentially all work relating to both diagnosis and vaccination concerned this particular species. Thus, it is not clear to what extent diagnostic procedures developed and vaccination approaches taken would also detect or protect against other species such as Candida glabrata. 
Therefore, there is a need in the art to identify and isolate novel stress proteins and nucleic acids encoding the same from Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus, which are useful in the detection, diagnosis and treatment of infections caused by these organisms.
The present invention provides methods and compositions comprising isolated nucleic acid molecules specific to Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus, as well as vector constructs and isolated polypeptides specific to Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus. Such compositions and methods are useful for the diagnosis of infection and for generating an immune response to the respective organisms.
Thus, in one aspect the present invention provides an isolated nucleic acid molecule encoding a Neisseria meningitidis Hsp70. In some embodiments, the isolated nucleotide molecule is selected from the group consisting of: (a) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:1 (FIG. 4); (b) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:1 from nucleotides 358-2286; (c) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:2 (FIG. 6) from nucleotides 4-1932; (d) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:3 (FIG. 8); (e) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:4 (FIG. 9); (f) an isolated nucleic acid molecule complementary to any one of the nucleotides of SEQ ID NOS: 1 to 4 set forth in (a) through (e), respectively.
In another aspect, the present invention provides an isolated nucleic acid molecule which is a variant of, or is substantially similar to, the Neisseria Hsp70 nucleotide molecules described above. In further aspects the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is identical to a segment of contiguous nucleotide bases comprising at least 25% of SEQ ID NOS: 1 to 4 or a complement thereof or an isolated nucleic acid molecule encoding Hsp70 comprising a nucleic acid sequence that encodes a polypeptide comprising any one of SEQ ID NOS: 2, 3, or 4 or a variant Hsp70 that is at least 95% homologous to a polypeptide according to any one of SEQ ID NOS: 2, 3, or 4.
In one embodiment, the present invention provides an isolated nucleic acid molecule as described above, the molecule encoding a polypeptide that is able to be selectively bound by an antibody specific for a Neisseria meningitidis Hsp70.
In still another aspect, the present invention provides an isolated nucleic acid molecule encoding at least 8 contiguous amino acids of a Neisseria meningitis Hsp70 polypeptide selected from amino acid residues of FIG. 6, wherein the encoded Neisseria meningitidis Hsp70 polypeptide is able to bind to a major histocompatibility complex.
In still further aspects the present invention provides an isolated Neisseria meningitidis Hsp70 polypeptide.
In some embodiments, the isolated Hsp70 polypeptide comprises the amino acid sequence of FIG. 6, or variants thereof, preferably wherein the polypeptide is able to be selectively bound by an antibody specific for a Neisseria meningitidis Hsp70. In further embodiments, the isolated Hsp70 polypeptide is fused to an additional polypeptide to create a fusion protein.
In still yet further aspects the present invention provides an isolated Hsp70 polypeptide comprising at least 8 contiguous amino acids selected from amino acid residues of FIG. 6, wherein the Hsp70 polypeptide is capable of binding to a major histocompatibility complex and eliciting or enhancing an immune response to Neisseria meningitidis in a human being.
In certain embodiments, the isolated Hsp70 polypeptide is derived by proteolytic cleavage or chemical synthesis, or is an expression product of a transformed host cell containing a nucleic acid molecule encoding the Hsp70 or portion thereof. In further certain embodiments, the isolated Hsp70 polypeptide comprises greater than 95% homology to the Hsp70 polypeptide of FIG. 6, and the isolated Hsp70 polypeptide is able to be selectively bound by an antibody specific for a Neisseria meningitidis Hsp70.
In still yet another aspect the present invention provides an isolated polypeptide wherein the polypeptide is an expression product of a transformed host cell containing one of the aforementioned nucleic acid molecules derived from Neisseria.
In still yet further aspects the present invention provides vectors comprising at least one of the aforementioned nucleic acid molecules derived from Neisseria. In certain embodiments, the vector is an expression vector comprising a promoter in operative linkage with the isolated nucleic acid molecule encoding the Hsp70 or portion thereof, preferably further comprising a selectable or identifiable marker and/or wherein the promoter is a constitutive or an inducible promoter. The present invention also provides host cells containing such vectors. In certain embodiments, the host cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell, a plant cell and an insect cell.
In still yet other aspects the present invention provides compositions comprising a Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition is suitable for systemic administration, oral administration, intranasal administration or parenteral administration.
In yet other aspects the present invention provides methods for eliciting or enhancing an immune response in a mammal against Neisseria, comprising administering to the mammal in an amount effective to elicit or enhance the response, a Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; methods for eliciting or enhancing an immune response in a mammal to a polypeptide comprising administering to the mammal a fusion protein containing sequences of the polypeptide fused to the Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; and methods for eliciting or enhancing an immune response in a mammal against a target antigen comprising administering to the mammal the target antigen joined to a Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent.
In still another aspect, this invention provides PCR primers and probes for detecting DNA encoding a Neisseria meningitidis. Hsp70 that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 1-4, or to compliment thereof. In a related aspect, the invention provides a method for diagnosing the presence of a Neisseria meningitidis in a subject sample that includes the steps of obtaining a DNA fraction from the subject sample; and performing a PCR amplification of the DNA fraction using at least one PCR primer that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 1-4, or a compliment thereof.
The present invention also provides an isolated nucleic acid molecule encoding a Aspergillus fumigatus Hsp60. In some embodiments, the isolated nucleotide molecule is selected from the group consisting of: (a) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:5 (FIG. 14); (b) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:5 from nucleotides 300-2234; (c) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:5 from nucleotides 300-410, nucleotides 514-655 and nucleotides 724-2234; (d) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:6 (FIG. 16); (e) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:7 (FIG. 18); (f) an isolated nucleic acid molecule complementary to any one of the nucleotides of SEQ ID NOS: 5 to 7 set forth in (a) through (e), respectively.
In another aspect, the present invention provides an isolated nucleic acid molecule that is a variant of, or is substantially similar to, the Aspergillus Hsp60 nucleotide molecules described above. In further aspects, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is identical to a segment of contiguous nucleotide bases comprising at least 25% of SEQ ID NOS: 5 to 7, or a complement thereof, or an isolated nucleic acid molecule encoding Hsp60 and comprising a nucleic acid sequence of any one of SEQ ID NOS: 5, 6 or 7 or a variant Hsp60 that is at least 95% homologous to a nucleic acid sequence comprising any one of SEQ ID NOS: 5, 6, or 7.
In one embodiment, the present invention provides an isolated nucleic acid molecule according as described above, the molecule encoding a polypeptide that is able to be selectively bound by an antibody specific for a Aspergillus fumigatus Hsp60.
In still another aspect in one aspect the present invention provides an isolated nucleic acid molecule encoding at least 8 contiguous amino acids of a Aspergillus fumigatus Hsp60 polypeptide selected from amino acid residues according to FIG. 14, wherein the encoded Aspergillus fumigatus Hsp60 polypeptide is able to bind to a major histocompatibility complex.
In still further aspects the present invention provides an isolated Aspergillus fumigatus Hsp60 polypeptide.
In some embodiments, the isolated Hsp60 polypeptide comprises the amino acid sequence of FIG. 14, or variants thereof, preferably wherein the polypeptide is able to be selectively bound by an antibody specific for a Aspergillus fumigatus Hsp60. In further embodiments, the isolated Hsp60 polypeptide is fused to an additional polypeptide to create a fusion protein.
In still yet further aspects the present invention provides an isolated Hsp60 polypeptide comprising at least 8 contiguous amino acids selected from amino acid residues according to FIG. 14, wherein the Hsp60 polypeptide is capable of binding to a major histocompatibility complex and eliciting or enhancing an immune response to Aspergillus fumigatus in a human being.
In certain embodiments, the isolated Hsp60 polypeptide is derived from proteolytic cleavage or chemical synthesis, or is an expression product of a transformed host cell containing a nucleic acid molecule encoding the Hsp60 or portion thereof. In certain further embodiments, the isolated Hsp60 polypeptide comprises greater than 95% homology to the Hsp60 polypeptide of FIG. 14, and the isolated Hsp60 polypeptide is able to be selectively bound by an antibody specific for a Aspergillus fumigatus Hsp60.
In still yet another aspect the present invention provides an isolated polypeptide wherein the polypeptide is an expression product of a transformed host cell containing at least one of the nucleic acid molecules derived from the aforementioned Aspergillus molecules.
In still yet further aspects the present invention provides vectors comprising at least one of the aforementioned nucleic acid molecules derived from Aspergillus. In certain embodiments, the vector is an expression vector comprising a promoter in operative linkage with the isolated nucleic acid molecule encoding the Hsp60 or portion thereof, preferably further comprising a selectable or identifiable marker and/or wherein the promoter is a constitutive or an inducible promoter. The present invention also provides host cells containing such vectors. In certain embodiments, the host cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell, a plant cell and an insect cell.
In still yet other aspects the present invention provides compositions comprising an Aspergillus Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition is suitable for systemic administration, oral administration, intranasal administration or parenteral administration.
In yet other aspects the present invention provides methods for eliciting or enhancing an immune response in a mammal against Aspergillus, comprising administering to the mammal in an amount effective to elicit or enhance the response, an Aspergilllus Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; methods for eliciting or enhancing an immune response in a mammal to a polypeptide comprising administering to the mammal a fusion protein containing sequences of the polypeptide fused to the Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; and methods for eliciting or enhancing an immune response in a mammal against a target antigen comprising administering to the mammal the target antigen joined to an Aspergillus Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent.
In still another aspect, this invention provides PCR primers and probes for detecting DNA encoding a Aspergillus fumigatus Hsp60 that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 5-7, or to compliment thereof. In a related aspect, the invention provides a method for diagnosing the presence of a Aspergillus fumigatus in a subject sample that includes the steps of obtaining a DNA fraction from the subject sample; and performing a PCR amplification of the DNA fraction using at least one PCR primer that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 5-7, or a compliment thereof.
The present invention further provides an isolated nucleic acid molecule encoding a Candida glabrata Hsp60. In some embodiments, the isolated nucleotide molecule is selected from the group consisting of: (a) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:8 (FIG. 21); (b) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:8 from nucleotides 258-1964; (c) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:9 (FIG. 23); (d) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:10 (FIG. 25); (e) an isolated nucleic acid molecule complementary to any one of the nucleotides of SEQ ID NOS: 8 to 10 set forth in (a) through (d), respectively.
In another aspect, the present invention provides an isolated nucleic acid molecule which is a variant of, or is substantially similar to, the Candida Hsp60 nucleotide molecules described above. In further aspects the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is identical to a segment of contiguous nucleotide bases comprising at least 25% of SEQ ID NOS: 8 to 10 or a complement thereof or an isolated nucleic acid molecule encoding Hsp60 comprising a nucleic acid sequence that encodes a polypeptide comprising any one of the polypeptides according to FIG. 21, 23 or 25, or a variant Hsp60 that is at least 95% homologous to a polypeptide according to any one of FIG. 21, 23 or 25.
In one embodiment, the present invention provides an isolated nucleic acid molecule according as described above, the molecule encoding a polypeptide that is able to be selectively bound by an antibody specific for a Candida glabrata Hsp60.
In still another aspect in one aspect the present invention provides an isolated nucleic acid molecule encoding at least 8 contiguous amino acids of a Candida glabrata Hsp60 polypeptide selected from amino acid residues according to FIG. 21, wherein the encoded Candida glabrata Hsp60 polypeptide is able to bind to a major histocompatibility complex.
In still further aspects the present invention provides an isolated Candida glabrata Hsp60 polypeptide.
In some embodiments, the isolated Hsp60 polypeptide comprises the amino acid sequence of FIG. 21, or variants thereof, preferably wherein the polypeptide is able to be selectively bound by an antibody specific for a Candida glabrata Hsp60. In further embodiments, the isolated Hsp60 polypeptide is fused to an additional polypeptide to create a fusion protein.
In still yet further aspects the present invention provides an isolated Hsp60 polypeptide comprising at least 8 contiguous amino acids selected from amino acid residues according to FIG. 21, wherein the Hsp60 polypeptide is capable of binding to a major histocompatibility complex and eliciting or enhancing an immune response to Candida glabrata in a human being.
In certain embodiments, the isolated Hsp60 polypeptide is derived from proteolytic cleavage or chemical synthesis, or is an expression product of a transformed host cell containing a nucleic acid molecule encoding the Hsp60 or portion thereof. In further certain embodiments, the isolated Hsp60 polypeptide comprises greater than 95% homology to the Hsp60 polypeptide of FIG. 21, and the isolated Hsp60 polypeptide is able to be selectively bound by an antibody specific for a Candida glabrata Hsp60.
In still yet another aspect the present invention provides an isolated polypeptide wherein the polypeptide is an expression product of a transformed host cell containing at least one of the aforementioned nucleic acid molecules derived from Candida.
In still yet further aspects the present invention provides vectors comprising at least one of the aforementioned nucleic acid molecules derived from Candida. In certain embodiments, the vector is an expression vector comprising a promoter in operative linkage with the isolated nucleic acid molecule encoding the Hsp60 or portion thereof, preferably further comprising a selectable or identifiable marker and/or wherein the promoter is a constitutive or an inducible promoter. The present invention also provides host cells containing such vectors. In certain embodiments, the host cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell, a plant cell and an insect cell.
In still yet other aspects the present invention provides compositions comprising a Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition is suitable for systemic administration, oral administration, intranasal administration or parenteral administration.
In yet other aspects the present invention provides methods for eliciting or enhancing an immune response in a mammal against Candida, comprising administering to the mammal in an amount effective to elicit or enhance the response, a Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; methods for eliciting or enhancing an immune response in a mammal to a polypeptide comprising administering to the mammal a fusion protein containing sequences of the polypeptide fused to the Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; and methods for eliciting or enhancing an immune response in a mammal against a target antigen comprising administering to the mammal the target antigen joined to a Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent.
In still another aspect, this invention provides PCR primers and probes for detecting DNA encoding a Candida glabrata Hsp60 that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 8-10, or to compliment thereof. In a related aspect, the invention provides a method for diagnosing the presence of Candida glabrata in a subject sample that includes the steps of obtaining a DNA fraction from the subject sample; and performing a PCR amplification of the DNA fraction using at least one PCR primer that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 8-10, or a compliment thereof.
These and other aspects of the present invention will become evident upon reference to the present specification and the attached drawings. In addition, various references are set forth herein that describe in more detail certain procedures or compositions (e.g., plasmids, etc.); all such references are incorporated herein in their entirety by reference.